TGM6 L517W Is Not a Pathogenic Variant for Spinocerebellar Ataxia Type 35

ARTICLE OPEN ACCESS TGM6 L517W is not a pathogenic variant for spinocerebellar ataxia type 35

Yanxing Chen, MD, PhD, Dengchang Wu, MD, PhD, Benyan Luo, MD, PhD, Guohua Zhao, MD, PhD, and Correspondence Kang Wang, MD, PhD Dr. Zhao [email protected] Neurol Genet 2020;6:e424. doi:10.1212/NXG.0000000000000424 or Dr. Wang [email protected] Abstract Objective To investigate the pathogenicity of the TGM6 variant for spinocerebellar ataxia 35 (SCA35), which was previously reported to be caused by pathogenic mutations in the gene TGM6.

Methods Neurologic assessment and brain MRI were performed to provide detailed description of the phenotype. Whole-exome sequencing and dynamic mutation analysis were performed to identify the genotype.

Results The proband, presenting with myoclonic epilepsy, cognitive decline, and ataxia, harbored both the TGM6 p.L517W variant and expanded CAG repeats in gene ATN1. Further analysis of the other living family members in this pedigree revealed that the CAG repeat number was expanded in all the patients and within normal range in all the unaﬀected family members. However, the TGM6 p.L517W variant was absent in 2 aﬀected family members, but present in 3 healthy individuals.

Conclusions The nonsegregation of the TGM6 variant with phenotype does not support this variant as the disease-causing gene in this pedigree, questioning the pathogenicity of TGM6 in SCA35.

From the Department of Neurology (Y.C., G.Z.), the Second Affiliated Hospital, School of Medicine, Zhejiang University; and Department of Neurology (D.W., B.L., K.W.), the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

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Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AD = autosomal dominant; DRPLA = dentatorubral-pallidoluysian atrophy; GTCS = generalized tonic-clonic seizure; SCA = spinocerebellar ataxia.

Spinocerebellar ataxias (SCAs) are a group of autosomal members. Briefly, 22-FAM-labeled primer sets were used for dominant (AD) disorders that mainly affect the cerebellar triplet repeat primed PCR, followed by PCR product processed function. SCAs are genetically heterogeneous, and there are by capillary electrophoresis with the ABI3730xl (Applied Bio- more than 40 subtypes of SCAs and more than 30 genes systems, Foster, CA). The primer sequences of all SCAs genes identified responsible for them (neuromuscular.wustl.edu/). were designed using reference sequences from GenBank. Data SCA35 was reported to be associated with mutation in trans- were analyzed with GeneMapper v4.0 (Applied Biosystems). glutaminase 6 gene (TGM6), which codes for transglutaminase 6 protein (TG6) in 2010.1 A 4-generation Chinese AD-SCA Data availability family was identified to have 9 affected members showing The data that support the findings of this study are available progressive gait instability, scanning speech, and poor dexterity from the corresponding author on reasonable request. in hands. Linkage analysis suggested that the disease-causing gene was located on chromosome 20p13-12.2, and exome sequencing showed that the c.1550T>G (p.L517W) variant Results cosegregated with the phenotypes. Another variant c.980A>G The proband (IV:1, figure, A) is a 28-year-old woman, who fi 2 (D327G) was identi ed in another AD-SCA family. In the presented to the neurology outpatient clinic with a 13-year ff following years, another 13 variants were reported in di erent history of unprovoked seizures. She developed generalized ethnic groups including Chinese, Asian, European, and tonic-clonic seizures (GTCSs) at age 15 years, which mainly 2–9 Hispanic, mainly in Asia. Here, we report an AD family with occurred in the morning, on awakening. She was then pre- 6 patients presenting with ataxia. Nonsegregation of the TGM6 scribed with several antiepileptic drugs, including valproate, p.L517W variant with phenotype questions the pathogenicity lamotrigine, and levetiracetam, as monotherapy or polytherapy of this variant. with different combinations, but still experiencing 3–5GTCSs each year. At age 25 years, she started to develop myoclonic jerks, which manifested with a brief, shock-like, involuntary Methods movement of the upper limbs, more severe distally and aggra- fi Participants vated by motion, and clonazepam was added with signi cant The 4-generation pedigree was recruited from the First Af- improvement. One year ago, the patient gradually exhibited filiated Hospital of Zhejiang University School of Medicine. unsteadiness, speech disturbance, and memory decline. Neu- Neurologic examination was performed by at least 2 senior rologic examination revealed mild cerebellar dysarthria, dys- fi neurologists. metria performing nger-to-nose test, and positive Romberg sign. Ophthalmoplegia, nystagmus, and saccadic movements Standard protocol approvals, registrations, were not observed. She had normal muscle strength without and patient consents rigidity, normal deep tendon reflexes, and a Mini-Mental State This study was approved by the Medical Ethics Committee of Examination score of 20. Brain MRI revealed mild atrophy of the First Affiliated Hospital of Zhejiang University School of the cerebellum and cerebral cortex (figure). Video- Medicine (No. 2017-326). Written informed consent was electroencephalography (video-EEG) showed normal back- obtained from each participant or from a legal representative. ground activities and intermittent spikes involving the posterior head region. Photic sensitivity was absent. Considering the Genetic analysis progressiveness and the involvement of multiple neurologic Genomic DNA was extracted from the peripheral blood of systems of this patient, inherited neurodegenerative diseases family members. Exon-enriched DNA sequencing and bio- were suspected. Whole-exome sequencing identified a reported informatic analysis were performed on the Illumina HiSeq variant c.1550T>G (p.L517W) in the TGM6 gene. Given that X-ten (Illumina, CA) in high-output mode with 150 bp paired- epilepsy has not been reported in patients with TGM6 muta- end reads following the manufacturer’s instructions (Illumina) tions, we further studied ataxia caused by trinucleotide (or in the proband. The suspected pathogenic variants were vali- pentanucleotide) repeat expansion. Surprisingly, genetic testing dated by Sanger sequencing. The results of Sanger sequencing using capillary electrophoresis revealed 14/61 CAG repeats in performed in other living family members were used for the atrophin-1 (ATN1) gene on chromosome 12p13.31, which cosegregation analysis. A dedicated panel for screening SCAs is a well-established gene for DRPLA. However, this raises the including SCA1, 2, 3, 6, 7, 8, 10, 12, 17, and dentatorubral- question whether this patient happened to harbor double pallidoluysian atrophy (DRPLA) was used to test repeat mutations, which might have additive effects on the phenotype, expansions of related mutations in all the available family or the TGM6 p.L517W variant is not actually pathogenic.

2 Neurology: Genetics | Volume 6, Number 3 | June 2020 Neurology.org/NG Figure Family pedigree and brain MRI of the proband

(A) Family pedigree of DRPLA disease. (B) Brain MRI of the proband showing mild cerebellar atrophy. (C) Normal control of the TGM6 gene. (D) The TGM6 variant in the patient. Arrows indicate the variant site. DRPLA = dentatorubral-pallidoluysian atrophy.

We subsequently studied the whole family and found that unstable CAG trinucleotide repeat expansion in ATN1. Nor- family members I:2, II:1, II:3, III:1, and III:5 developed cere- mally, the CAG tract bears 6–35 repeats, which expands to over bellar ataxia and cognitive decline/dementia in their fifties or 49 in patients with DRPLA.10 The clinical features of DRPLA sixties (table). The TGM6 p.L517W variant was absent in are strikingly heterogeneous, depending on the age at disease patients III:5 and II:3, but present in unaffected individuals (II: onset and the prominent genetic anticipation.11 Patients with 4, III:3, and III:7). The nonsegregation of the TGM6 variant juvenile-onset (onset before age 20 years) frequently exhibit with phenotype does not support this variant as the disease- progressive myoclonic epilepsy, intellectual disabilities, and causing gene in this family. On the other hand, the CAG repeat ataxia. Various forms of seizures are common features in all number was expanded in all the patients (II:3, III:1, III:5, and patients with onset before age 20 years. Patients with onset IV:1) and within normal range in all the unaffected family after age 20 years typically present symptoms including cere- members with available DNA, perfectly cosegregating with bellar ataxia, choreoathetosis, and dementia. Therefore, the disease phenotype. No DNA was available from the deceased clinical manifestations of the proband comply with juvenile- patients (I:1, I:2, and II:1). Patient II:1 died of gastric cancer at onset DRPLA, while the other affected family members also fit age 53 years. The presence of the ATN1 variant in her children well the clinical spectrum of DRPLA. indicates that patient II:1 should be a carrier. This observation strongly suggests that the disease is caused by the CAG repeat The identification of TGM6 as a causative gene for SCA35 was expansion in ATN1 rather than the p.L517W variant in TMG6. first reported in 2010.1 The TGM6 p.L517W variant cosegregated with the phenotype in a Chinese 4-generation SCA family. Cosegregation of another variant of TGM6 with the Discussion phenotype was also identified in another 2-generation family. More variants in the TGM6 gene were later discovered in In the current study, we present a 4-generation pedigree with patients with ataxia manifestations by other groups, supporting ataxia and cognitive decline/dementia. One well-established the role of TG6 in ataxia syndrome.2,3,8 However, with the wide CAG expansion in ATN1 and another reported pathogenic application of the next-generation sequencing in clinical prac- TGM6 p.L517W variant were identified. Cosegregation anal- tice in recent years, increasing genetic results are available, ysis confirmed the ATN1 mutation as the disease-causing gene, which challenges the previously reported pathogenic variants casting doubts on the pathogenicity of the TGM6 p.L517W that have not been extensively validated. Besides, the emerging variant. public database-based variant analysis provides an accessible and efficient approach to test the pathogenicity probability of ATN1 is a well-established gene for DRPLA, which is a rare, suspected variants. Recently, in a Chinese exome sequencing inherited AD neurodegenerative disorder resulting from an cohort, 8 families were found to harbor the reported TGM6

Neurology.org/NG Neurology: Genetics | Volume 6, Number 3 | June 2020 3 Table Clinical information and genetic results in the family members

Family Age at c.1550T>G ATN1 CAG member Sex Age (y) onset (y) Clinical symptoms variant repeats

I:2 Female Deceased NA Ataxia / /

II:1 Female Deceased Fifties Ataxia / /

II:2 Male 83 / None Negative 14/17

II:3 Male 75 Sixties Ataxia/dementia Negative 9/54

II:4 Male 72 / None Positive 17/18

III:1 Male 56 52 Ataxia/cognitive Positive 17/55 decline

III:2 Female 50 / None Negative 14/18

III:3 Male 55 / None Negative 14/17

III:5 Female 53 48 Ataxia/cognitive Negative 14/56 decline

III:7 Male 51 / None Positive 17/17

IV:1 Female 28 15 Seizure/myoclonus/ Positive 14/61 ataxia/cognitive decline

Abbreviation: NA = not available.

variants but share no features of SCA35. They further reviewed provides direct and compelling evidence that the TMG6 the public database genomAD and found that the reported p.L517W is not pathogenic. pathogenic variants, including L517W, are significantly more common in East Asians than in other ethnic groups. Gene A few functional studies of the TMG6 variants indicate a bi- constraint metrics showed that both missense and loss-of- ologically possible link with neurodegeneration. The sub- function variants in TGM6 are unlikely to be disease causing. cellular distribution, expression, and in vitro activity of the 2 Inflation analysis of the reported pathogenic TGM6 variants variants of TGM6 (D327G and L517W) have been in- showed that there is an at least 111-fold inflation over disease vestigated. In 1 study, it was found that neither of the mutants prevalence of all AD SCAs. This level of inflation is far beyond changed the subcellular localization of TG6, but exhibited de- the threshold of 10-fold, which indicates a high chance of creased transglutaminase activity and exerted the cells more misdiagnosis/misclassification of the variant or very low pen- vulnerable to staurosporine-induced apoptosis.13 On the other etrance. They raised the concern that misclassification of be- hand, another study found that 5 previously reported TGM6 nign or low penetrant variants as pathogenic is a significant mutations, including L517W, were associated with nuclear problem, which would often result in genetic misdiagnosis.12 depletion of TG6 and loss of the transglutaminase activity, and However, this study still fails to reach a definite conclusion thus leading to the activation of the unfolded protein response regarding the causality between the genotype and phenotype. and neuronal death.8 These studies showed an increased vul- First, the inability to perform cosegregation analysis in this nerability of cells transfected with TGM6 variants, suggesting study cannot rule out the likelihood of incomplete penetrance a role of TG6 in neuronal viability. However, variants associ- of the variants. Second, the size of database and the ethnic ated biological function changes might not be responsible for recruiting bias should be taken into consideration when inter- the clinical manifestations observed in patients, which are also preting the results. Last, the majority of index individuals in commonly seen in risk genes. Even healthy individuals carry their cohort were infants or children, and half of their asymp- many rare protein-disrupting variants.14 Therefore, further tomatic variant–carrying parents were under middle age. Thus, functional studies that can replicate the disease-relevant phe- there exists possibility that they might become symptomatic at notypes are needed to establish the causal relationship between older age, given that the mean age at onset for SCA35 was 43.7 TGM6 variants and the disease phenotype. years.1 Pedigree cosegregation analysis could provide addi- tional evidence on the pathogenicity of the variants, especially We report a pedigree with ataxia, in which a well-established that lack of segregation of a variant is strong evidence against ATN1 mutation and a TGM6 variant were identified. Non- pathogenicity. Therefore, the pedigree we report here is of segregation of the TGM6 L517W variant with disease phe- special value. The nonsegregation of the variant in this pedigree notype indicates that this variant is not disease causing. We do

4 Neurology: Genetics | Volume 6, Number 3 | June 2020 Neurology.org/NG not rule out the possibility that certain genuine pathogenic TGM6 variants might exist. Appendix (continued)

Name Location Contributions Acknowledgment The authors thank the patients and their families for Guohua Department of Neurology, Designed and Zhao, MD, the Second Affiliated conceptualized the study; participating in this study. PhD Hospital, School of major role in acquisition Medicine, Zhejiang and interpretation of data; Study funding University, Hangzhou, and critical review China This study was supported by the National Key R&D Program of China (2017YFC0907700), National Natural Science Kang Wang, Department of Neurology, Designed and MD, PhD the First Affiliated Hospital, conceptualized the study; Foundation of China (81870826), and Zhejiang Provincial School of Medicine, major role in acquisition Natural Science Foundation of China (LY19H180006, Hangzhou, China and interpretation of data; and critical review LY18H090004).

Disclosure References Y. Chen, D. Wu, B. Luo, G. Zhao, and K. Wang report no 1. Wang JL, Yang X, Xia K, et al. TGM6 identified as a novel causative gene of spino- disclosures. Go to Neurology.org/NG for full disclosures. cerebellar ataxias using exome sequencing. Brain 2010;133:3510–3518. 2. Li M, Pang SY, Song Y, Kung MH, Ho SL, Sham PC. Whole exome sequencing identifies a novel mutation in the transglutaminase 6 gene for spinocerebellar ataxia in Publication history a Chinese family. Clin Genet 2013;83:269–273. fi 3. Pan LL, Huang YM, Wang M, et al. Positional cloning and next-generation se- Received by Neurology: Genetics November 16, 2019. Accepted in nal quencing identified a TGM6 mutation in a large Chinese pedigree with acute myeloid form March 13, 2020. leukaemia. Eur J Hum Genet 2015;23:218–223. 4. Guo YC, Lin JJ, Liao YC, Tsai PC, Lee YC, Soong BW. Spinocerebellar ataxia 35: novel mutations in TGM6 with clinical and genetic characterization. Neurology 2014; 83:1554–1561. 5. Fasano A, Hodaie M, Munhoz RP, Rohani M. SCA 35 presenting as isolated treatment- resistant dystonic hand tremor. Parkinsonism Relat Disord 2017;37:118–119. Appendix Authors 6. Yang ZH, Shi MM, Liu YT, et al. TGM6 gene mutations in undiagnosed cerebellar ataxia patients. Parkinsonism Relat Disord 2018;46:84–86. Name Location Contributions 7. Choi KD, Kim JS, Kim HJ, et al. Genetic variants associated with episodic ataxia in Korea. Sci Rep 2017;7:13855. Yanxing Department of Neurology, Drafted the manuscript; 8. Tripathy D, Vignoli B, Ramesh N, et al. Mutations in TGM6 induce the unfolded Chen, MD, the Second Affiliated major role in acquisition protein response in SCA35. Hum Mol Genet 2017;26:3749–3762. PhD Hospital, School of and interpretation of data; 9. Lin CC, Gan SR, Gupta D, Alaedini A, Green PH, Kuo SH. Hispanic spinocerebellar Medicine, Zhejiang and critical review ataxia type 35 (SCA35) with a novel frameshift mutation. Cerebellum 2019;18:291–294. University, Hangzhou, 10. Wardle M, Morris HR, Robertson NP. Clinical and genetic characteristics of non- China Asian dentatorubral-pallidoluysian atrophy: a systematic review. Mov Disord 2009; 24:1636–1640. Dengchang Department of Neurology, Acquisition and 11. Tsuji S. Dentatorubral-pallidoluysian atrophy. Handb Clin Neurol 2012;103: Wu, MD, the First Affiliated Hospital, interpretation of data and 587–594. PhD School of Medicine, critical review 12. Fung JLF, Tsang MHY, Leung GKC, et al. A significant inflation in TGM6 genetic risk Hangzhou, China casts doubt in its causation in spinocerebellar ataxia type 35. Parkinsonism Relat Disord 2019;63:42–45. Benyan Department of Neurology, Acquisition and 13. Guan WJ, Wang JL, Liu YT, et al. Spinocerebellar ataxia type 35 (SCA35)-associated Luo, MD, the First Affiliated Hospital, interpretation of data and transglutaminase 6 mutants sensitize cells to apoptosis. Biochem Biophys Res PhD School of Medicine, critical review Commun 2013;430:780–786. Hangzhou, China 14. Tennessen JA, Bigham AW, O’Connor TD, et al. Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 2012;337:64–69.

Neurology.org/NG Neurology: Genetics | Volume 6, Number 3 | June 2020 5 TGM6 L517W is not a pathogenic variant for spinocerebellar ataxia type 35 Yanxing Chen, Dengchang Wu, Benyan Luo, et al. Neurol Genet 2020;6; DOI 10.1212/NXG.0000000000000424

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References This article cites 14 articles, 1 of which you can access for free at: http://ng.neurology.org/content/6/3/e424.full.html##ref-list-1 Subspecialty Collections This article, along with others on similar topics, appears in the following collection(s): Dementia aphasia http://ng.neurology.org//cgi/collection/dementia_aphasia Gait disorders/ataxia http://ng.neurology.org//cgi/collection/gait_disorders_ataxia Myoclonus; see Movement Disorders/myoclonus http://ng.neurology.org//cgi/collection/myoclonus_see_movement_diso rders-myoclonus Spinocerebellar ataxia http://ng.neurology.org//cgi/collection/spinocerebellar_ataxia Trinucleotide repeat diseases http://ng.neurology.org//cgi/collection/trinucleotide_repeat_diseases Permissions & Licensing Information about reproducing this article in parts (figures,tables) or in its entirety can be found online at: http://ng.neurology.org/misc/about.xhtml#permissions Reprints Information about ordering reprints can be found online: http://ng.neurology.org/misc/addir.xhtml#reprintsus